Modulation of cell fates and activities by phthalazinediones

ABSTRACT

Phthalazinediones that function as intracellular redox modulators are useful in treating cells in various disease states where intracellular redox status is impaired. By buffering aberrant redox states, phthalazinediones enable cellular processes essential for survival and augment medical treatments. The phthalazinediones of the invention can modulate functions related to cell growth, differentiation, activity, or death, to correct aberrations and restore homeostasis, and can serve as adjunctive therapy in treating various disease conditions.

This application is a continuation-in-part of application Ser. No.10/283,647 filed Oct. 30, 2002, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Current medical treatments generally focus on the disease and strive toeliminate the inciting agent or the symptoms, often injuring healthytissue in the process. The present invention focuses instead on thepatient, to enable self-repair mechanisms by supporting the patient'sbody in controlling or stabilizing its cellular functions without toxicside effects. The methods and compositions of the invention comprisephthalazinedione compounds that buffer intracellular reduction andoxidation (redox) reactions and thereby modulate cellular functions ofgrowth, differentiation, activity, and death in various disease states.

In healthy cells, a balance of redox reactions maintains aphysiologically appropriate environment for various cellular functionsrelated to growth, differentiation, activity, and death. The propercoordination of such functions ensures homeostasis and the health ofcells. Research has shown that alterations in cellular redox statusaffect activities such as cellular signaling, suggesting that alteringthe cellular redox status could also affect cellular activation, whichresults from certain cellular signals (U.S. Pat. No. 5,994,402).Altering the intracellular redox state by depleting cells of glutathione(GSH), an endogenous “redox agent,” has also been shown to protect cellsfrom certain injury and to promote their survival (U.S. Pat. No.5,994,402), again suggesting a link between alterations in the cellularredox state and cellular functions.

Stresses that perturb a cell's redox status may be internal or external.For example, a genetic mutation may produce defective protein productsthat function abnormally or not at all. These defective proteins coulddisrupt certain cellular processes, including redox reactions. Cellularredox reactions may also be disrupted by microbes, toxins, allergens, orother agents external to the cell. The external stress could triggerdefensive responses that leave the cell's redox system depleted andunstable.

An imbalanced redox state, even if not the cause of a particular diseasecondition, may facilitate that condition by providing an “unhealthy”environment in which necessary cellular functions become impaired.Cellular redox status may become impaired in numerous diseaseconditions. Under the stress of a disease state, the rate of redoxreactions increases or decreases as needed by the cell. Significant orprolonged deviations in the intracellular redox status disable cellularprocesses, including defense mechanisms. When such cellular functionsare impaired, the survival of the cell becomes uncertain. Maintenance ofthe proper redox status is thus critical to the fate of the cell.

To counter and correct disturbances in the redox status, cells requireagents that can modulate redox imbalances, to facilitate reduction oroxidation reactions as appropriate. Agents currently available forcorrecting redox imbalances are inadequate in that they are labile,quickly oxidized, or unable to translocate to the proper region of thecell. Examples of such exogenous redox agents include cysteine, reducedlipoates or thiols, glucocorticoids, and other antioxidants. Redoxagents that remain stable, active, and functional in the cellularenvironment are necessary.

Although their role in modulating intracellular redox status was notrecognized, phthaloylhydrazide, phthalazinedione, and phthalazinederivatives have been described as having anti-inflammatory,anti-cancer, and anti-hypoxic effects (U.S. Pat. Nos. 6,686,347;6,489,326; 5,874,444; 5,543,410; 5,512,573; 4,250,180). However,toxicity and the lack of pharmacological activity of certainphthaloylhydrazides, including 2,3-dihydrophthalazine-1,4-dione and5-amino-2,3-dihydrophthalazine-1,4-dione, were noted (U.S. Pat. Nos.6,489,326; 5,543,410; 5,512,573). Luminol, also known aso-aminophthaloylhydrazide, 3-aminophthalhydrazide,5-aminophthaloylhydrazide, or 5-amino-2,3-dihydro-1,4-phthalazinedione,was considered toxic and used in photothermographic imaging,chemiluminescent assays and labeling of cellular structures, detectionof copper, iron, peroxides, or cyanides, and forensic science to detecttraces of blood (U.S. Pat. Nos. 5,279,940; 4,729,950; Merck Index, 13thed. (2001), monograph no. 5622).

Nonetheless, the compound 5-aminophthaloylhydrazide was identified foruse in treating inflammatory conditions such as ulcerative colitis,Crohn's disease, diffuse sclerosis, diarrhea, proctitis, hemorrhoids,anal fissures, dyspepsia, intestinal infection, Alzheimer's disease,osteoarthritis, macular degeneration, and proctosigmoiditis (U.S. Pat.Nos. 5,874,444; 5,543,410; EP 617024; RU2211036), as well as for use intreating psoriasis, infarct, and transplant rejection (U.S. Pat. Nos.6,489,326; 5,512,573). Other phthaloylhydrazide derivatives identifiedas having pharmacological activity include2,3-dihydrophthalazine-1,4-dione,2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt dihydrate,4-aminophthaloylhydrazide, 4,5-aminophthaloylhydrazide, and4,5-methylaminophthaloylhydrazide (U.S. Pat. Nos. 6,489,326; 5,512,573;RU 2113222). Phthalazinedione compounds, including luminol, have alsobeen described as an inhibitor of poly (ADP-ribose) polymerase, anenzyme that responds to DNA damage (U.S. Pat. Nos. 5,874,444; 5,719,151;5,633,282), and for treating conditions involving the functions of poly(ADP-ribose) polymerase (U.S. Pat. Nos. 5,874,444; 5,719,151;5,633,282). A method of manufacturing the sodium salt of5-amino-2,3-dihydrophthalazine-1,4-dione and its pharmaceutical use forimmunomodulation, inflammation, and anti-oxidant treatment have beendescribed (U.S. Pat. No. 6,489,326; RU 2222327).

SUMMARY OF THE INVENTION

Phthalazinediones of the invention may be used to modulate redoximbalances and to support a patient's body in a variety of diseasestates and in treating metabolic distress, inflammation, infectiousconditions, neurological disorders, immune disorders, proliferativediseases, and senescence. The phthalazinediones may also be used inconjunction with standard treatment methods such as chemotherapy,radiation, nutrition, pharmaceutical treatment, and surgery.

DETAILED DESCRIPTION

The present invention describes the use of phthalazinedione compounds intreating diseases or disorders involving impaired or aberrantintracellular redox states. By buffering redox imbalances,phthalazinediones can reversibly and selectively modulate cellularfunctions, e.g., upregulating mitochondrial aerobic metabolism when acell under stress needs energy for defense or repair, or downregulatingmetabolism when the stressed cell is overactive. Phthalazinediones canmodulate cellular processes such as proliferation, secretion,differentiation, transformation, migration, and apoptosis, without toxicside effects on healthy cells.

Under any stress, intracellular redox status is inevitably impaired asaerobic metabolism is necessarily overworked. Any stress to the cell,especially if prolonged, will deplete the cell of endogenous redoxagents, including thiols, glutathione, thioredoxins, iron-sulfurproteins, cysteine, and thiol proteins, as well as redox-sensitiveproteins such as catalase. Chronic stress leads to cellular andorganelle thiol deficiencies, as blood cysteine is limited. In turn,since many cellular pathways are controlled by or depend onintracellular redox activities, thiol deficiencies lead rapidly toimpaired energy production, with increased oxidant production andprogressive mitochondrial and cell death.

In mitochondrial aerobic metabolism, electron flow is fragile and easilyperturbed by oxidant stresses. Under stress, the cell must rapidlyincrease both the electron flow and the subsequent membrane proton (H⁺)gradient. However, electron flow and proton gradient may fail ifoveractivated or stressed. Electrons are then diverted directly tooxygen (O₂), producing toxic superoxide (O₂ ⁻), while the protongradient declines, hindering ATP production. Moreover, under oxidantstress, mitochondrial membrane channels and permeability pores becomeoxidized, which distorts the channels and opens the pores. Consequently,protons, substrate anions, glutamate, reductants, cytochrome c, andnucleotides all leak through the distorted channels and opened pores,leaving the mitochondrion and cell deficient in essential substances,energy, and redox status.

With prolonged thiol deficiencies, replacement therapy with availablethiols is difficult and usually inadequate. Cysteine and other reducedthiols are labile and rapidly oxidized to toxic metabolites in thepresence of oxygen. Most antioxidants, which dissipate oxygen-basedoxidants, are unable to penetrate to the electron-transporting innermitochondrial membrane to modulate the iron-sulfur protein mediatedelectron flow in mitochondrial Complex III or to stabilize disulfidecross-linkages that control permeability of the mitochondrial megaporesand channels. Antioxidants also cannot supply the cysteine required inthe manufacture of most proteins or the energy required to combatchronic stresses or repair cellular damages.

In general, a therapeutically effective amount of a phthalazinedione ofthe invention that is sufficient to ameliorate disease symptoms willdepend on the acuteness of the disease, the particular redox status ordeficiency of the patient, the developmental condition of the stressedcell, and also the state of oxidation of the phthalazinedione, but willbe in the range of about 0.01-100.0 mg per kg of body weight or about1.0-10,000.0 mg per day, e.g., administered in amounts of 1.0, 10.0,50.0, 100.0, 200.0, 300.0, 400.0, 500.0, 600.0, 700.0, 800.0, 900.0,1000.0, 2000.0, 3000.0, 4000.0, 5000.0, 6000.0, 7000.0, 8000.0, 9000.0,or 10,000.0 mg.

The phthalazinedione compounds of the present invention are preferablyincorporated into pharmaceutical forms suitable for administration byoral, nasal, mucosal, vaginal, rectal, transdermal, or parenteralroutes, including subcutaneous, intramuscular, intravenous, andintraperitoneal, e.g., tablet, capsule, granule, powder, solution,suspension, microsphere, liposome, colloid, lyophilized composition,gel, lotion, ointment, cream, spray, and suppository, and preferablyinclude pharmaceutically acceptable excipients, carriers, adjuvants,diluents, or stabilizers as is well known to the skilled in the art.

The phthalazinedione may be a derivative compound containing asubstituent that enhances the activity, stability, or other property ofthe compound. Such a derivative compound may be an aminophthalazinedione or a phthalazinedione comprising a haloamino,alkylamino, acylamino, alkanolamino, alkenylamino, alkoxyamino,haloalkylamino, alkylamino, or sulfhydrylamino (thiolamino ormercaptoamino) group or other substituents that confer a preferredfunction on the compound. Furthermore, the phthalazinedione may be abromoamino, chloroamino, fluoroamino, iodoamino, methylamino,ethylamino, propylamino, isopropylamino, methanoylamino (formylamino),ethanoylamino (acetylamino), propanoylamino, hydroxylamino,carboxylamino, methanolamino, ethanolamino, propanolamino,methenylamino, ethenylamino, propenylamino, methoxyamino, ethoxyamino,propoxyamino, or dimethylamino derivative.

Examples of such phthalazinedione derivatives include, but are notlimited to, 5-amino-2,3-dihydrophthalazine-1,4-dione (luminol),6-amino-2,3-dihydrophthalazine-1,4-dione (isoluminol),5-amino-2,3-dihydrophthalazine-1,4-dion-8-yl (luminyl),N-bromo-5-amino-2,3-dihydrophthalazine-1,4-dione,N-chloro-5-amino-2,3-dihydrophthalazine-1,4-dione,N-fluoro-5-amino-2,3-dihydrophthalazine-1,4-dione,N-iodo-5-amino-2,3-dihydrophthalazine-1,4-dione,N-methyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-ethyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-propyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-isopropyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-methanoyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-ethanoyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-propanoyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-hydroxyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-carboxyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-methanol-5-amino-2,3-dihydrophthalazine-1,4-dione,N-ethanol-5-amino-2,3-dihydrophthalazine-1,4-dione,N-propanol-5-amino-2,3-dihydrophthalazine-1,4-dione,N-methenyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-ethenyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-propenyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-methoxy-5-amino-2,3-dihydrophthalazine-1,4-dione,N-ethoxy-5-amino-2,3-dihydrophthalazine-1,4-dione,N-propoxy-5-amino-2,3-dihydrophthalazine-1,4-dione,N,N-dimethyl-5-amino-2,3-dihydrophthalazine-1,4-dione,N-acetylcysteine-5-amino-2,3-dihydrophthalazine-1,4-dione, andN-acetylglutathione-5-amino-2,3-dihydrophthalazine-1,4-dione.Enantiomers, isomers, tautomers, esters, amides, salts, solvates,hydrates, analogues, metabolites, free bases, or prodrugs of thephthalazinedione or its derivative are also contemplated by theinvention.

In an embodiment of the invention, phthalazinediones can be used toeither facilitate or inhibit electron flow in mitochondria, and thuscontrol ATP production. For example, in vitro, at the low dose of 20-50μM, amino phthalazinediones facilitate electron flow at mitochondrialComplex III, thereby increasing ATP production, DNA synthesis, and cellcycling, for cell growth. At an intermediate dose of 100 μM, aminophthalazinediones slow down electron flow, with concomitant effects onATP production, DNA synthesis, and cell cycling, so that differentiationcan proceed. At the high dose of 200 μM, amino phthalazinedionescompletely stop ATP production, DNA synthesis, and cell cycling in thestressed cell, such that the cell becomes quiescent but does not die.

Thus, phthalazinediones of the invention may be used to control cellfates and serve as redox buffers for the redox- and thiol-sensitiveenergy producing pathways in the mitochondrion, signaling pathways atthe cell plasma membrane, and glutamate uptake and cytokine secretion byastrocytes in the central nervous system (Trotti et al., J. Biol. Chem.271: 5976-5979, 1996). In particular, amino phthalazinediones catalyzedisulfide cross-linkages in the adenine nucleotide translocase (ANT) ofthe mitochondrial anion channels and in the megapores, which preventsenergy production, increases production of the potent signal transducershydrogen peroxide (H₂O₂) and superoxide (O₂ ⁻) (Zamzami et al., Oncogene16: 1055-1063, 1998; Constantini et al., J. Biol. Chem. 271: 6746-6751,1996), and liberates the apoptosis-inducing factors cytochrome c andAIF.

Under certain conditions, loss of redox control may cause:

(1) cross-linking of thiols in the adenine nucleotide translocase andother proteins, which then opens the mitochondrial transmembrane poresand channels and leads to a decline in mitochondrial voltage and energyproduction (Constantini et al., J. Biol. Chem. 271: 6746-6751, 1996;Larochette et al., Exp. Cell Res. 249: 413-421, 1999; Zanzami et al.,Oncogene 16: 1055, 1998);

(2) increases in intracellular calcium levels;

(3) activation of redox defenses and heat shock proteins;

(4) activation of redox-sensitive cell cycling factor AP-1 and E2F/Rbpathway;

(5) activation of apoptotic pathways via AsK-1, with liberation ofcaspases, cytochrome c, and AIF from the failing mitochondrion;

(6) a decline in ADP-dependent electron flow, as well as alteration ofmobility of redox sensitive iron-sulfur proteins at mitochondrialComplex III (Zhang et al., J. Biol. Chem. 275: 7656-7662, 2000);

(7) oxidation of macromolecules, including redox-sensitive proteins suchas glutarnate transporters (Trotti et al., J. Biol. Chem. 271:5976-5979, 1996), mitochondrial DNA, and membrane lipids;

(8) a failure in modulation of redox-sensitive phosphatases PTB-1,SHP-1, and SHP-2 (Doza et al., Oncogene 17: 19-26, 1998); and

(9) dysregulation of the thiol-sensitive MAP kinase-Ras pathway, whichcontrols cellular proliferation.

With redox support to buffer the redox stress and restore the redoxstatus, the mitochondrion resumes energy production. The cell thenrepairs stress-induced damages, restocks essential substrates, andremoves all offenders, in essence treating its own disease. To besuccessful, any exogenous redox agent must therefore enable the cell tocorrect the redox aberration, remove the cellular stress, and repairmechanical damages, without toxic side effects. Accordingly, in anembodiment of the invention, phthalazinediones primarily supportmetabolically distressed cells in a subject, by buffering theintracellular redox status without toxic side effects, to enable thesubject's cellular repair or defense functions, rather than treat aparticular condition in terms of trying to eliminate the disease or itscause.

Redox support therapy may be utilized in various disease states, as in:

(1) conditions of metabolic distress, such as redox imbalance ordeficiency, metabolic syndrome (Syndrome X), intoxication, diabetes,insulin resistance, hyperglycemia, hypoglycemia, hyperinsulinemia,hypoinsulinemia, hypoadiponectinemia, hyper fatty acidemia,inflammation, tissue injury, and burns;

(2) inflammatory conditions where overactive cells, e.g., lymphocytes,macrophages, astrocytes, or microglia, strain redox defenses and energyproduction, such as Parkinson's disease, Alzheimer's disease,Huntington's disease, multiple sclerosis (MS), Guillain-Barré syndrome(GBS, acute inflammatory demyelinating polyneuropathy, acute idiopathicpolyradiculneuritis, acute idiopathic polyneuritis, or Landry'sascending paralysis), Lyme disease, Crohn's disease, ulcer, colitis,hemorrhoids, diarrhea, proctitis, arthritis, osteoarthritis, rheumatoidarthritis, stroke, myocardial infarction, auricular or atrialfibrillation, preexcitation syndrome (Wolff-Parkinson-White syndrome),arteriosclerosis, atherosclerosis, inflammation of blood vessels thatcharacterize vascular disease in heart and brain, thromboangiitisobliterans (Winiwarter-Buerger disease), other inflammatory conditionsof the vascular system, inflammatory conditions of the skin such asdermatitis, eczema, psoriasis, postoperative complications, peritonitis,bronchitis, and pleurisy;

(3) infectious conditions such as HIV infection, acquiredimmunodeficiency disease (AIDS), hepatitis, herpes, Lyme disease, toxicshock syndrome, dysentery, erysipelas, hantavirus pulmonary syndrome,respiratory syndromes such as pneumonia and tuberculosis, and otherviral or bacterial related conditions or diseases;

(4) neurological disorders such as Alzheimer's disease, Parkinson'sdisease, Huntington's disease, Cockayne syndrome, amyotrophic lateralsclerosis (ALS or Lou Gehrig's disease), MS, Bloom's disease, dementia,dystonia, Charcot-Marie-Tooth syndrome (CMT), Dejerine-Sottas syndrome,Roussy-Levy syndrome, Rosenberg Chutorian syndrome, Korsakoff syndrome,Friedreich ataxia, Machado-Joseph disorder, progressive supranuclearpalsy (PSP or Steele-Richardson-Olszewski syndrome), GBS, neurallymediated hypotension, pain syndromes such as fibromyalgia, reflexsympathetic dystrophy syndrome (RSDS or complex regional pain syndrome,CRPS), myofascial pain syndrome (MPS), patellofemoral pain syndrome, andother neurodegenerative conditions;

(5) immune disorders, including multiple chemical sensitivity syndrome(MCS), leukemia, GBS, immune deficiency diseases such as AIDS,transplant or graft rejection as in graft-vs-host disease, allergies orallergic reactions, sinusitis or sinus conditions, eczema, psoriasis,asthma, autoimmune diseases or disorders such as systemic lupuserythematosus, scleroderma, and rheumatoid arthritis, Wegener'sgranulomatosis, diabetes mellitus, Crohn's disease, and Wiskott-Aldrichsyndrome;

(6) proliferative diseases, such as cancer, including leukemia,lymphoma, and myeloma, tumors, melanoma, carcinoma, sarcoma, prostatichypertrophy or adenoma, atherosclerosis, angiogenesis, restenosis,proliferative diseases of the vascular system or endometrium, and othersyndromes of rapid cell proliferation or clonal expansion; and

(7) senescence, such as that linked to or caused by geneticabnormalities as in Down syndrome, trichothiodystrophy, ataxiatelangiectasia (AT), Bloom's disease, xeroderma pigmentosum, and p53overactivity, and other premature aging or wasting diseases such asmuscular dystrophy, age-related macular degeneration (AMD), metabolicsyndrome, and aging.

To survive any stress, cells must replace depleted thiols and maintainoptimum mitochondrial redox potentials and activities. In one embodimentof the invention, therapy includes combined treatment withphthalazinediones and compounds to replace the lost thiols, oxidativelyprotect the phthalazinedione, eliminate the source of stress, orotherwise support the subject in fighting a particular condition. Acompound that is an amino acid, antibiotic, antiviral agent,antiinflammatory agent, antioxidant, immunomodulator, reductant,oxidative protector, steroid, or vitamin may be beneficial. Compoundssuch as a cysteine (e.g., acetyl cysteine, N-acetylcysteineamide),glutathione, lipoic acid (e.g., alpha lipoic acid, dehydrolipoic acid),hydralazine, thioredoxin, biopterin (e.g., tetrahydropterin,sepiapterin), glucocorticoid, dexamethasone, rasagiline, ferulic acid,minocyline, menadione, tetracycline, isosorbate dinitrate,dextromethorphan, or mixtures thereof may be used. The additionalcompound may be administered simultaneously, separately, orsequentially.

The preferred active ingredients may be formulated into a pharmaceuticalcomposition with one or more pharmaceutically acceptable excipients. Forexample, a pharmaceutical composition may comprise a phthalazinedione, aglutathione, and one or more pharmaceutically acceptable excipients. Thepharmaceutical composition may be in the form of a tablet, capsule,granule, powder, solution, suspension, microsphere, liposome, colloid,lyophilized composition, gel, lotion, ointment, cream, spray, orsuppository and administered intravenously, intramuscularly,intraperitoneally, subcutaneously, orally, nasally, mucosally,transdermally, parenterally, vaginally, or rectally. A therapeuticallyeffective amount of the phthalazinedione or a pharmaceutical compositioncomprising a therapeutically effective amount of the phthalazinedione isadministered to a subject in metabolic distress, to maintain the desiredredox status and mitochondrial energy production, as well as theredox-sensitive MAP kinase-Ras PT3K signal transduction pathways.

The amount of phthalazinedione needed or effective at any one point iscell- and stress-dependent. Optimum dosage and treatment require properdiagnosis of the thiol redox status of the patient's aerobic metabolismin the stressed mitochondria. Administration sufficiently early on incell or stress development, such that cellular structures or functionshave not deteriorated beyond repair, e.g., mitochondria swollen andleaky, cells entering apoptosis, would be particularly beneficial. Thethiol redox status must also be frequently monitored, sincephthalazinediones can be oxidatively very labile and rapidly expended.

In tissue culture, small doses of less than 1 μg/ml of an aminophthalazinedione are effective for conditions with chronic losses ofcells, especially of stem or developing cells, as inneuroimmunodegenerative syndromes. In conditions where proliferation andapoptotic rates are out of control, including cancer, autoimmunity,infection, and traumas, doses greater than 50 μg/ml of aminophthalazinediones are required. Successful treatment with thephthalazinedione compounds of the invention therefore depends both onredox diagnosis with repeated assessment of cellular thiol redox statusand on maintenance of proper dosage of the phthalazinedione over time.Treatment with phthalazinediones is directed at cells or organs in whichstress has dysregulated thiol redox homeostasis, with resulting energydeprivation and oxidant stress.

In one embodiment of the invention, amino phthalazinediones also act asefficient substrates for reaction with many of the reactive oxygenspecies and radicals that are inevitably generated in the stressedmitochondrion. Because of their antioxidant, anti-inflammatory,antiproliferative, immunomodulatory, redox-buffering, and non-toxicproperties, phthalazinediones can be beneficial as adjunctive supporttherapy for the stressed cell regardless of the compromising stress orits downstream symptoms. In rare disease states, redox support may besufficient for the diseased cell to treat itself, but in somesituations, the cell will also need the mechanical, pharmacological, orgenetic support of standard medical treatments such as radiation,chemotherapy, laser therapy, surgery, medication, and nutrition used intreating particular disease conditions. As adjunct support therapy, thephthalazinediones of the invention may be administered simultaneously,separately, or sequentially for a combined treatment regimen. Thefollowing examples further illustrate the invention.

EXAMPLE 1 Uncontrolled Inflammation

In inflammatory conditions, such as acute infections, wounds, and immuneresponses, phthalazinediones, especially amino phthalazinediones,quickly ameliorate the painful redox-induced edematous swelling andfacilitate rapid healing. Edematous inflammatory lesions in intestines,such as duodenal ulcers, ulcerative colitis, and acute vascular injury,are all suppressed to some degree by thiol redox modulators, includingdihydrolipoates, reduced biopterins, amino phthalazinediones, and moreslowly by glucocorticoids. Healing rates increase, with replacement ofthe injured epithelial cells by thiol redox-stimulated new cell growth.Thus, phthalazinediones, acting as thiol redox modulators, suppressinjurious over-reactive inflammatory responses and also facilitatehealing and replacement of injured cells.

EXAMPLE 2 Uncontrolled Proteolysis

In conditions with aberrant or uncontrolled proteolysis, as in apoptosisor necrosis, thiol redox modulators, especially thioredoxin, eitherupregulate or downregulate the regulatory proteases involved inprocessing and digesting the thiol redox dependent caspases,endonucleases, and histone deacetylases responsible for protein and DNAhydrolysis. Diamide, a phthalazinedione with activity similar to theoxidized 4-amino phthalazinedione, can activate and cross-link proteasesthat hydrolyze procaspase 3 to the active caspase fragments that, alongwith cytochrome c, initiate the apoptotic cascade in the nucleus.

Since these cross-linking agents can also oxidize essential membraneproteins, such as the adenine nucleotide translocase in mitochondria oramyloid protein fragments in brain, the result is membrane poreformation in mitochondria with increased reactive oxygen species andcell destruction (Ueda et al., J. Immunol. 161: 6689-6695, 1998). Thus,reduced phthalazinediones can up- or down-regulate redox-sensitiveproteases and thereby dictate life and death of stressed proliferatingcells.

EXAMPLE 3 Helicase Deficiencies

The XPD gene of the xeroderma pigmentosum family codes for a helicase.In XPD deficiency, DNA transcription and repair functions are impaired,resulting in multiple symptoms of early aging (De Boer et al., Science296: 1276-1281, 2002). Wasting, loss of subcutaneous fat and musclecells, gray brittle greasy hair with hyperplasia of sebaceous andmammary glands, severe osteoporosis, atrophic germ and stem cells, andimmunoneurodegenerative and hyperplastic changes all occur prematurelyin XPD-deficient mice or humans.

The failure to maintain normal numbers of cells or normal amounts ofcellular thiols, at least in the brittle hair, suggests that a globalthiol redox deficiency is responsible for the progressive wasting andchronic cell losses. Since amino derivative phthalazinediones withreduced thiol redox modulators, at low dosage, stimulate cell growth andmaintain thiol redox status in cells, treatment with appropriate amountsof reduced thiol redox modulators combined with the phthalazinediones ofthe present invention will likely prevent the premature aging inregulatory gene deficiencies such as XPD deficiency.

EXAMPLE 4 p53 and Aging

In conditions where cell growth and tumor formation are constantlysuppressed by growth suppressor genes like p53, signs of premature agingand replication senescence appear early (Tyner et al., Nature 415:45-50, 2002). Chronic cell losses in skin, hair, bone, adipose tissue,and the immune system occur. The p53 protein is a potent transcriptionfactor that suppresses cell growth and DNA synthesis and is also anactivator of genes that induce oxidative stress and apoptosis, such asBax and caspases 3 and 9.

Thiol redox modulators such as phthalazinediones, which maintaincellular replication pathways by modulating cellular redox status,override the p53-induced suppression and maintain a balance betweenapoptotic or proliferation pathways, depending on dosage. Data tosupport this homeostatic concept for thiol redox modulators inp53-induced aging are under evaluation. Since thiol redox modulatorsbeneficially balance rates of cell death and proliferation in othersyndromes of premature aging, including XPD deficiency andretrovirus-induced degenerative diseases, it is likely that thiol redoxmodulators, at appropriate dosages, can re-balance the p53-induced thiolredox potential and thereby prevent the degenerative sequelae.

EXAMPLE 5 Retroviral-Induced Redox Imbalance

Oncogenic retroviral infections such as HIV in humans or MOMU-LV-Ts1 inmice cause degenerative changes with severe losses of brain cells,immune cells, and germ cells. Other cells like astrocytes and microgliain brain become activated, secrete nitric oxide (NO) and superoxide (O₂⁻), and grow and accumulate excessively. This imbalance in cell growthand death rates eventually leads to fatal immune and neuronal deficiencysyndromes with subsequent transformation in some cells.

In mice infected at 2 days of age with the Ts1 virus, hind limbparalysis occurs with severe wasting, especially of immune organs. Inhumans infected with HIV, severe immune deficiency with sensory andmotor neuropathy also results. In these wasting syndromes withdisordered life and death pathways in various cells, some therapeuticattempts with thiol redox modulators other than amino phthalazinedioneshave been partially successful (Lynn and Wong, Neuroimmunomodulation 4:277-284, 1997; Yan et al., FASEB J. 15: 1132-1138, 2001). In thesestudies, phthalazinediones plus thiol redox modulators appear to besufficient to maintain survival and an adequate intracellular thiolredox potential in brain and in thymus.

Since retroviruses activate caspase-dependent apoptosis, and since thiolredox modulators, oxidized and reduced, regulate caspase production fromprocaspases (Nobel et al., Chem. Res. Toxicol. 10: 636-643, 1997), thiolredox modulators in mitochondria, especially an amino phthalazinedioneplus dexamethasone, will likely prevent both the loss and thehyperplasia of cells dysregulated by the viruses. Experiments usingvarious thiol redox modulator regimens as preventative therapy inTs1-infected mice are currently underway.

EXAMPLE 6 Polyglutamine Model

In disease states where aberrant peptides slowly accumulate in thebrain, early neuronal death with glial hypertrophy occurs. InHuntington's disease, polyglutamine sequences or tracts accumulate inthe huntingtin protein. These tracts bind to and inhibit transcriptioncomplexes containing Sp-1 and TAFII130 coactivators. Transcription ratesdecrease, and dysregulated neurons slowly die, first in the caudatenucleus and later in the hippocampus.

Non-proliferating, non-replaceable neurons usually die from metabolicredox imbalances, rather than from programmed death. Therefore, neuronaldeath in Huntington's disease is likely to be redox-mediated and inducedby activation of redox-sensitive cytokines, metalloproteases, andreactive oxygen species (ROS) by activated migroglia and astrocytes(Chen et al., Nature Med. 6: 797-801, 2000). In that case, reduced thiolredox-modulators, which at low doses promote cell growth and longevityin redox-suppressed cells, should prove to be useful therapy (Dunah etal., Science 296: 2238-2243, 2002).

In other neurodegenerative syndromes in which aberrant peptidesaccumulate, including Alzheimer's and Parkinson's diseases, presenilinor synucleins may be responsible for accumulation of the Lewy bodies andβ-amyloid peptides. Accumulation of these hydrophobic peptides inplasma, mitochondrial, or endoplasmic reticulum membranes of the cellmay be responsible for the neuronal losses in these syndromes. Thesetoxic peptides, like the polyglutamine proteins in Huntington's disease,also lead to astroglia-induced imbalances in thiol redox metabolism,with cell swelling, membrane leakiness, and mitochondrial necrosis.Maintenance of thiol redox status with reduced thiol redox modulators,especially an amino phthalazinedione and acetyl cysteine, should preventor delay the neuronal death in these degenerative diseases (Wolfe andSelkoe, Science 296: 2156-2157, 2002; Welhofen et al., Science 296:2215-2218, 2002).

EXAMPLE 7 NMDA-Induced Excitotoxicity Model

In NMDA-induced neuronal excitotoxicity, secreted microglialinflammatory products—glutamate, quinolinic acid, inflammatorycytokines, tumor necrosis factor, IL-1B, superoxide (O₂ ⁻), and nitricoxide (NO)—are likely responsible for the neuronal necrosis (Tikka andKolstinaho, J. Immunol. 166: 7527-7533, 2001). These excitotoxins allrapidly perturb redox homeostasis in neurons, which slowly die, and inactivated astroglia, which become activated and proliferate.

Minocycline, a cyclic polyhydroxy ketonic amide, which suppressesmitochondrial activity, prevents both the NMDA-induced proliferation ofand toxic secretions by activated astrocytes, as well as the subsequentneuronal death (Tikka and Kolstinaho, J. Immunol. 166: 7527-7533, 2001).This suggests that cell death in neurons, secretory proliferativeactivation of astroglia, and proliferative response in astrocytes in thespinal cord are mitochondrial redox-mediated and that correction ofthiol redox status by phthalazinediones should be able to control thefate of these brain cells.

EXAMPLE 8 Premature Aging with Cancer Models

In regulatory gene-dependent syndromes of premature aging, includingataxia telangiectasia, Down syndrome, trichothiodystrophy, Bloom'sdisease, p53 over-activity (De Boer et al., Science 296: 1276-1281,2002; Tyner et al., Nature 415: 45-50, 2002), in which life and death ofspecific cell types are aberrant, appropriate treatments in vitro and invivo with thiol redox modulators have been partially successful. Inataxia telangiectasia gene (ATM) deficiency in mice, early pretreatmentwith dexamethasone, the glutathione secretagogue, completely preventsthe excessive proliferation and development of the fatal thymic cancer.

Other thiol redox modulators, such as N-acetyl cysteine anddehydrolipoic acid, also delay the premature degeneration of cells andthe thymomas. Thiol redox modulators also correct the delayeddifferentiation and excessive production of DNA in ATM-deficientlymphoid cells (Yan et al., FASEB J. 15: 1132-1138, 2001; Lynn et al.,unpublished). However, in the ATM-deficient mice, treatment was fullysuccessful only if the thiol redox modulators were applied early, beforetwo weeks of age and before tumor development.

Dexamethasone alone completely prevents tumor formation if given to10-day old ATM-deficient mice for three weeks, but does not suppresstumor growth or increase longevity if given at physiologic doses atthree months of age. Whether amino phthalazinediones with other thiolredox modulators, which suppress growth of non-transformed ATM-deficientcells in vitro, can fully suppress tumors in vivo, without toxicity, hasnot been rigorously evaluated. Cross-linking redox modulators such asdiamide, menadione, and oxidized phthalazines are known to stop cellgrowth, activate caspases, and initiate apoptosis in some tumor cells(Pias and Aw, FASEB J. 16: 781-790, 2002).

EXAMPLE 9 Oxygen-Based Model

In acute metabolic distress, as in hypoxia, redox-sensitivetranscription factors such as H1FA are rapidly activated, orunder-activated if the oxygen deprivation is not too severe. Thesetranscription factors are triggered by the alternate redox-sensitivemammalian target of rapamycin (mTOR) signal transduction pathway, whichis upregulated by low oxygen, ATP, and amino acids. Activated mTORmarkedly upregulates DNA synthesis and cellular proliferation,especially in endothelial and vascular smooth muscle cells.Consequently, mTOR is involved in many redox-sensitive proliferativediseases of vascular tissues, including diabetic retinopathy, psoriasis,rheumatoid arthritis, certain tumors, and arteriosclerosis (Humar, FASEBJ. 16: 771-780, 2002).

Whether mTOR or its upstream activators are redox sensitive is notclear. Nonetheless, oxygen at low dose, like amino phthalazinediones atlow dose, increases proliferation, whereas oxygen at very low dose(<1%), or phthalazines at high dose, stop proliferation and activatecell death pathways. Vascular cell fates are clearly dependent onexternal redox agents that modulate internal redox status, and theresponses and fates of these cells are readily controlled in adose-dependent manner by external redox agents such as oxygen, aminophthalazinedione, diamide, or permeant thiols, which modulate themTOR-signaling pathway. These redox agents should therefore be useful asredox buffers in controlling the redox-sensitive mTOR pathway,ameliorating various vascular proliferative inflammatory diseases, andcontrolling angiogenesis both in tumor growth and inflammatorysyndromes, particularly in brain.

EXAMPLE 10 Uncontrolled Oxygen Models

In uncontrolled oxygen metabolism, oxygen is not fully reduced, suchthat reactive oxygen intermediates accumulate. Cell fate is highlydependent on the concentration, location, and longevity of reactiveoxygen species such as O₂ ⁻, H₂O₂, OH., NO, and OHOO.. In proliferatingvascular smooth muscle cells, addition of O₂ ⁻ or H₂O₂ quickly increasesDNA synthesis, via activation of the Id3/E2F pathway. In the presence ofiron plus H₂O₂, which produces the more potent OH. radical, DNAsynthesis, Id3 protein, and Id3 mRNA rapidly decline, while cell deathrates increase. Thus, the fate of growing smooth muscle cells is highlydependent on oxygen redox status.

The two oxygen redox-sensitive genes, Id3 and GKLF, which aredifferentially responsive to oxygen redox status, are most sensitive torapid changes in concentrations of reactive oxygen species. Withincreased concentrations in OH., Id3 expression is downregulated, GKLFexpression is upregulated, and DNA synthesis ceases (Nickenig et al.,FASEB J. 16: 1077-1086, 2002). The GKLF protein, when oxidized, isactivated and inhibits Id3 expression by binding to the Id3 promoter.The Id3 protein, when reduced, is activated and upregulates theE2F-controlled proliferation pathway.

Thus, oxygen redox status, like thiol redox status, is a potentregulator of cell fates. Moreover, the two redox pathways, and the twoelectron acceptors oxygen and sulfur, interact repeatedly. For example,reduced phthalazines or thiols chemically reduce most of the reactiveoxygen species, including peroxynitrite (ONOO⁻). Tetrahydropterin (BH₄),a major cellular reductant in the central nervous system, reducesreactive oxygen species and the inducible oxidase iNOS. Under redoxstress, in the presence of tetrahydropterin, iNOS produces nitric oxide(NO). Under redox stress when tetrahydropterin or reduced thiols arelimited, iNOS produces superoxide (O₂ ⁻). In turn, superoxide (O₂ ⁻) orhydrogen peroxide (H₂O₂) activates Id3 and the E2F-controlled DNAsynthesis pathway but only in the absence of iron or copper (Dehmer etal., J. Neurochem. 74: 2213-2216, 2000; Husman et al., FASEB J. 10:1135-1141, 2002; Liberatore et al., Nature Med. 5: 1403-1409, 1999).

Thus, intracellular redox homeostasis, whether oxygen or thiol-mediated,is dependent on concentrations of cellular reductants—tetrahydropterin,glutathione, cysteine, NADPH—and cellular oxidants—O₂ ⁻, H₂O₂, NO, OH.,Fe³⁺—as well as on concentrations of permeant extracellularreductants—reduced thiols, tetracyclines, phthalazines—and permeantextracellular oxidants—O₂ ⁻, gamma radiation, doxorubicin,glucocorticoids, cis-platinum, doxirubicin, etc. Consequently, redoxhomeostasis can be readily maintained by appropriate doses of permeantredox agents, notably by phthalazinediones, and with protean therapeuticimplications. Phthalazines, tetracyclines, or thiols (Tikka andKolstinaho, J. Immunol. 166: 7527-7533, 2001) potentially dictate andcontrol the cell fate in activated or stressed cells, whether thedisease-inducing redox imbalance is oxygen- or sulfur-mediated. Inaddition to controlling proliferation and activation pathways, theseredox modulators also scavenge destructive oxygen radicals and therebyprevent apoptotic and necrotic pathways.

Potential therapeutic usefulness of these redox modulators in astrogliainduced neurodegenerative diseases (Tikka and Kolstinaho, J. Immunol.166: 7527-7533, 2001), in renal allografts (Husman et al., FASEB J. 10:1135-1141, 2002), and in inflammation-induced cell damages (Ryan et al.,Curr. Opinion in Rheumatology 8: 238-247, 1996) are now beingrecognized. Thus, redox modulating compounds, especiallyphthalazinediones, that modulate both the oxygen and sulfur redoxpathways are proving to be therapeutically useful in situations wherethe patient's redox mechanisms are out of control.

EXAMPLE 11 Chronic Inflammation Model with Accumulation of Excess Lipids

In situations where foreign fats such as oxidized fatty acids orcholesterol accumulate, a chronic inflammatory reaction ensues.Signaling and transport processes in lipid-laden membranes falter.Lipid-laden activated macrophages accumulate. Oxidant stress follows,due to deficiency in glucose transport in the lipid-laden membranes andthe increased production of oxidants and proteases by the influx ofactivated macrophages. Chronic localized abscesses form. In vasculartissue, atherosclerosis with occlusive diseases, stroke, myocardialinfarction, cystic mastitis, wet macular degeneration, and engorgedactivated adipocytes are the result. In all these syndromes, thiol redoxhomeostasis becomes gravely perturbed and cellular redox damage occurs.Metabolic syndrome, or Syndrome X, with insulin resistance is an earlysequela.

Therapies known to modulate the above lipid- and redox-induced syndromesinclude:

(1) thiol redox modulators, especially amino phthalazinediones, tobuffer the aberrant thiol redox status;

(2) anti-proteases, especially minocycline, to block the excessproteolytic activity and suppress O₂ ⁻ production by the induced NOsynthase by macrophages;

(3) peroxisome proliferators, to accelerate oxidation of accumulatinglipids;

(4) caloric restriction, to block input and accumulation of the aberrantlipids and O₂ ⁻;

(5) glucocorticoids, to deplete thiols by excretion, inhibit growth, andaccelerate death of the overactivated macrophages and microglia; and

(6) sepiapterin, to prevent superoxide (O₂ ⁻) production by iNOS in thebrain and to prevent activation of the apoptosis stimulating kinaseAsK-1, especially in the brain.

Many external therapies are therefore available to modulate and preventthe chronic abscess formations induced by accumulation of aberrantoxidized fats in cell membranes. To fully maintain optimum redox status,over time, in disease states with differing etiologies, variouscombinations and doses of all six redox approaches may be required. Withoptimum redox support, the subject will repair most damages and inducethe means—for example, peroxisome proliferator receptors (PPARs) andadiponectin—to remove the offending fats. In severe defects, specificanti-proteases and antioxidants as those listed above are essential foroptimal therapy.

EXAMPLE 12 Redox-Controlled Neuronal Survival

Oxidizing agents such as H₂O₂, NMDA agonists, and N-nitrosoguanidinesrapidly kill primary neurons. In the presence of oxidants theredox-sensitive nuclear poly (ADP-ribose) polymerase, which cleaves NAD⁺to ADP-ribose and stabilizes nuclear proteins by ADP-ribosylating them,is rapidly activated. This depletes the neuron of NAD⁺ as well as thereductants NADH and NADPH. This also rapidly facilitates nuclear uptakeof the mitochondrial redox-sensitive flavoprotein, apoptosis inducingfactor (AIF).

These oxidants also open the redox-sensitive permeability transitionpores and anion channels in mitochondrial membranes, which release AIF.AIF is then taken up by the poly (ADP-ribose) polymerase-activatednucleus to initiate chromatin condensation. Chromatin condenses,mitochondria grow swollen, and mitochondrial processes become uncoupled.Mitochondria then produce more oxidants, O₂ ⁻ and H₂O₂, and produce lessATP. In addition, the oxidants rapidly induce reshuffling of plasmamembrane ionic phospholipids with surface exposure of phosphatidylserine. This rapidly alters permeability and transport activities inplasma, mitochondrial, endoplasmic reticulum, and nuclear membranes. Theplasma and endoplasmic reticulum membranes leak calcium, which activatesinnumerable signal transduction pathways, including ATM, mTOR, and p38MAPK (Yu et al., Science 297: 259-263, 2002; De Giorgi et al., FASEB J.10: 607-609, 2002).

Thus, redox status of most cellular membranes is rapidly altered bybrief exposure to permeant oxidants, and cell death rapidly ensues,through both apoptotic (nuclear) and necrotic (plasma membrane) changes.Membrane-permeant reductants, such as phthalazinediones plus reducedbiopterins and thiols, should be able to buffer and maintain the properredox status in membranes of oxidant-stressed organelles as occurs inacute neurodegenerative syndromes such as hypoxia or glucose deficientstates, or in chronic inflammatory states such as Parkinson's disease,Alzheimer's disease, ALS, MS, AT, or aging.

EXAMPLE 13 Role of Thiol Redox Status in Mitochondrial Activities

The major source of chemical energy and heat in aerobic cells ismitochondria. The modulatable permeable pores and channels inmitochondria are exquisitely sensitive to thiol redox status. Thespecific mitochondrial channel is composed of two thiol redox sensitiveproteins located in the inner membrane—adenine nucleotide translocase(ANT) and voltage dependent anion channel (VDAC)—and other coproteinssuch as cyclophilin D, hexokinase, benzodiazepine receptors, and theBcl-2/Bax family of peptides. These proteins together control thepermeability and transport of mitochondrial transmembrane channels andpores, which control ADP entry, proton exit, electron flow,intracellular calcium concentration, and O₂ ⁻ production.

Bax, benzodiazepine receptors, and hexokinases, which bind to the outermembrane of mitochondria, regulate transport and pore formation in thesemembranes. Major physiologic modulators of this mitochondrialtransmembrane pore include:

(1) transmembrane voltage, which is generated by electron and protongradients;

(2) inducible membrane proteins, Bc1-2 and Bax; and

(3) thiol redox status, the redox state of Cys-56 on the channel proteinANT being a major regulator of the permeability of mitochondrialtransmembrane pores (He and Lemasters, FEBS Letters 512: 1-7, 2002).

Thiol oxidants or cross-linking agents such as diamide or diethylmaleate distort and open mitochondrial pores and channels, and uncoupleelectron flow, allowing oxygen to trap electrons and produce O₂ ⁻, H₂O₂,and other radicals. Energy production declines, and mitochondria releasecytochrome c, caspases, and AIF. Destructive cytochrome c,redox-sensitive proteases, and caspases are activated in the cytoplasmand the nucleus, causing cell death, both apoptotic and necrotic.

Reduced thiols, dithiothreatol, glutathione, N-acetyl cysteine, oragents such as Bcl-2, Bongkrekic acid, cyclosporine A, or chaperonecyclophilins that can stabilize ANT sulfhydryls and maintain porepermeability status can completely prevent the electron leak and thecell death (Armstrong and Jones, FASEB J. 16: [online], Jun. 7, 2002;Castantini et al., Oncogene 19: 307-314, 2000; Hong et al., FASEB J. 16:1633-1636, 2002). Whether permeant reductants such as thephthalazinediones of the present invention, which stabilize and maintainthiol redox status, alone stabilize the thiol redox sensitivemitochondrial transmembrane pore in vivo is currently underinvestigation.

Under oxidant stressed conditions, including radiation, chemotherapy,occlusive vascular diseases, leptin-deficient or resistin-inducedobesity, caloric excesses, and type II diabetes, in which optimal thiolredox status is not maintained by the diseased adipose tissue of thepatient, therapeutic support by external thiol redox buffers will beacutely necessary, at least until the patient can repair and buffer thestressed and imbalanced thiol redox status and fully activate itshypoxia-inducible transcription factors (Wenger, FASEB J. 16: 1151-1162,2002; De Giorgi et al., FASEB J. 10: 607-609, 2002).

Depending on the type of oxidative stress, labile vicinal cysteinylresidues on ANT undergo cyclic oxidation, ionization, and eventuallycross-linking. These oxidations and cross-linkages of protein thiolsgreatly perturb channel functions, especially by thiol cross-linkingcyclic amines, diazenes (diamide), or phenylarsines. Uptake of ADPfails, protons are released with collapse of the inner membranepotential, ordered electron flow at mitochondrial Complex III falters,and O₂ now accepts the fluxing electrons with production of O₂ ⁻ andother radicals. The oxidant-producing mitochondria release cytochrome cand AIF, and downstream oxidation of NF_(κ)B, AP1 (major transcriptionfactor for proliferation), AsK-1 (apoptosis stimulating kinase),glutathione, Bax, HDAC (histone deacetylase in nucleus), PTEN(phosphatase in cytoplasm), and ATM occurs. Apoptosis, senescence,quiescence, or necrosis results, depending largely on the extent andduration of the redox stress. A photoactive diamine fluorescent cation,tetramethyl-rhodamine, which accumulates in mitochondria and releasesfree radicals when photoactivated, is a potent agonist of themitochondrial transmembrane pore. When tetramethyl-rhodamine isactivated, all downstream effects of oxidation and cross-linking ofANT's labile cysteinyl residues occur, including translocation andpolymerization of Bax in mitochondrial membranes. These effects arefully inhibited by Bongkrekic acid, a specific inhibitor ofmitochondrial transmembrane pores (De Giorgi et al., FASEB J. 10:607-609, 2002), as well as by reduced thiols, reduced phthalazines,cyclophilins, and pterines. These observations suggest that the fate ofcells under stress is largely dictated by mitochondrial thiol redoxstatus, and that cell fates are readily buffered or controlled bypermeant lipophilic redox-sensitive amines, such as phthalazinediones,tetrahydrobiopterin, and permeant thiols.

EXAMPLE 14 Thiol Redox Status in Mitochondria in Cancer Treatment

Controlling entry and exit of small molecules—Ca²⁺, H⁺, O₂ ⁻ andsubstrate anions through the redox- and voltage-sensitive mitochondrialchannels and pores is to control cell fates. These channels and poresmodulate concentrations of intracellular cations Ca²⁺ and H⁻,intracellular anions ADP, ATP, malate, and glutamate, and intracellularthiols, glutathione, cysteine, thioredoxin, and biopterin. By thesemeans, these channels can indirectly modulate redox-sensitive sites insignal transduction, proliferation, development, transcription,apoptosis pathways, and necrosis pathways, thereby dictating cell fates.

Many agents that can directly modulate these pores are in use forantiproliferative therapies, notably as treatments forhyperproliferative syndromes and cancer (Miccoli et al., J. Nat. CancerInst. 90: 1401-1406, 1998; Ravagnan et al., Oncogene 18: 2537-2546,1999; Larochette et al., Exp. Cell Res. 249: 413-471, 1999). Three broadclasses of modulators are in use—lipophilic peptides, lipophilic amines,and thiol redox-reactive cyclic amines.

Lipophilic peptides are useful as antiproliferative andanti-inflammatory therapies. These peptides, primarily Bax, Bcl-2, andcyclosporine A, either block or bypass mitochondrial transmembranechannels by creating pores of oxidized polymerized peptides of variablepermeability in the mitochondrial outer membranes (De Giorgi et al.,FASEB J. 10: 607-609, 2002). The redox-insensitive lipophilic benzoamines are useful in cancer therapy. Diazepam and lonidamine, forexample, bind to mitochondrial benzodiazepine receptors in themitochondrial matrix, block mitochondrial electron flow and ATPsynthesis, and induce apoptotic and necrotic death in rapidly growingcells (Miccoli et al., J. Nat. Cancer Inst. 90: 1401-1406, 1998). As forthe thiol redox-sensitive cyclic amines, their usefulness inmitochondrial transmembrane pore modulation has not been fully explored.

Diamide (diazenedicarboxylic acid), the thiol cross-linking non-cyclicamine, completely opens mitochondrial transmembrane pores, which causesthe mitochondrial transmembrane potential to collapse, with dissipationof H⁺ (pH) gradients, production of O₂ ⁻, and release of the apoptosisinducing factors cytochrome c and AIF. Consequently, cells slowly diedepending on their supplies of reduced thiols, primarily glutathione(Zamzami et al., Oncogene 16: 1055-1062, 1998). However, although apotent eradicator of cancer cells and other proliferating cells of thesubject, this cross-linking non-cyclic amine is too toxic for clinicaluses.

Other cyclic lipophilic amines, such as amino phthalazinediones,biopterins, and rhodamines, which accumulate electrostatically inmitochondrial transmembrane pores and accept and release both electronsand protons, reversibly serve as both electron and pH buffers in thepolarized channels and pores. In this manner, the ionic and oxidativestatus of the labile sulfhydryl in ANT is maintained by these redox- andpH-sensitive amines. The cyclic amines thus affect voltage in thechannels, and fluxing electrons are either trapped by O₂ as O₂ ⁻ orproceed downstream with production of H₂O and ATP. At low doses of thesecompounds, electron flow increases, electrons proceed downstream to H₂O,ATP production increases, DNA synthesis and cell proliferation increase,and cell death is aborted. At high doses, electron flow to H₂Odecreases, substrate anion translocations falter, membrane potentialdeclines, ATP production ceases, as does electron flow, and cells gointo a quiescent G₀/G₁ phase or apoptosis.

With the lipophilic tetramethyl-rhodamine, many electrons are shunteddirectly to O₂, with the result that O₂ accumulates, mitochondrialtransmembrane pores open with loss of membrane potential, and apoptoticand necrotic pathways are activated (De Giorgi et al., FASEB J. 10:607-609, 2002). Phthalazinediones, such as amino derivatives, combinedwith reduced biopterins, thiols, or lipoic acid, modulate electron flowto O₂ ⁻ or H₂O (Lynn et al., unpublished). Specifically, at low doses,amino phthalazinediones upregulate the subject's immune responses toeradicate cancerous cells. At high doses, amino phthalazinediones stopproliferation of hyperproliferating cancerous cells. Thus, byupregulating or downregulating particular cells, amino phthalazinedionesare useful in cancer treatment (Tzyb et al., Int. J.Immunorehabilitation 12: 398-403, 1999).

Modulation of mitochondria by these bifunctional cyclic phthalazines ismost effective in controlling cell fate in proliferating cells that aredeficient in biological thiol redox buffers (Armstrong and Jones, FASEBJ. 16: [online], Jun. 7, 2002; Larochette et al., Exp. Cell Res. 249:413-471, 1999), or in proliferating cells deficient in cell cyclecheckpoint genes (Yan et al., Genes and Dev., in press). Thus, redox-and pH-sensitive amines that buffer by dually modulating mitochondrialtransmembrane pores and anion channels are clinically useful both inpreventing and treating hyperproliferation states such as cancers.

EXAMPLE 15 Use of Phthalazinediones in Chronic Dys-Metabolic Syndromes

Food intake, especially fat, with excess deposition of fat in adiposecells causes production and secretion of large amounts of the adiposetissue defense peptide hormones—resistin, leptin, tumor necrosis factor,adiponectin. These collagen- and complement-like peptides facilitateuptake of glucose and combustion of long-chain fatty acids viaperoxisome proliferator receptors (PPAR) and mitochondria, withproduction of heat in the muscle mitochondria, facilitated by activatinguncoupling proteins in mitochondria. This removal of the excess fattyacids relieves the fatty acid-induced stress in adipocytes and alsolowers levels of toxic, free fatty acids in blood.

However, in time, with prolonged intake of fatty foods, as in affluentsocieties, and with consequent excessive storage of fat in adiposecells, these overstuffed fat cells produce and secrete more of theinflammatory cytokines, tumor necrosis factor, and resistin (aredox-sensitive adipokine), at the expense of secretion of adiponectin.In aging individuals with overstuffed fat cells, blood levels of tumornecrosis factor and resistin are high; adiponectin andplasminogen-activator inhibitors are low; glucose, free fatty acids,triglyceride, and insulin are high; and the PPARγ/RXR (retinoid Xreceptor) complexes in fat and muscle cells are under-activated.Vascular accidents in heart and brain, with atherosclerotic plaques, arealso greatly increased in these insulin-resistant individuals. Thismetabolic syndrome, also called Syndrome X, is epidemic.

Metabolic syndrome is a condition marked by excessive abdominal fat,diabetes, high blood pressure, and cholesterol problems, and is causedby the body's inability to use insulin efficiently, which in turnresults from overeating and inactivity. Metabolic syndrome is currentlyand partially treated with various benzolated thiazolidinediones. Thesecyclic nitrogenous diketones, which are structurally similar to thephthalazinediones of the present invention, bind to the promoters ofPPARγ in the nucleus and activate multiple gene families that activateperoxisomal fatty acid oxidation with increased production ofadiponectin and catalase, increased glucose uptake, and increasedproduction of enzymes required for fatty acid synthesis and oxidationand for terminal differentiation in adipocytic precursor cells. At highconcentrations, these diketone ligands of PPARγ also block proliferationand activities of activated macrophages, endothelial cells, microglia inbrain, and probably proliferating smooth muscle cells in atheromatousplaques. Thus, benzolated thiazolidinediones are useful in preventingmetabolic syndrome and its downstream sequelae, including insulinresistance, vascular degeneration with hypertension, macrophageproliferation and hyperactivity, with plaque formation and type IIdiabetes.

Benzolated phthalazinediones chemically resemble benzolatedthiazolidinediones and are known to reproduce some functions ofbenzolated thiazolidinediones, perhaps as a ligand for PPARγ. Inparticular, amino phthalazinediones, like benzolated thiazolidinediones,also stop proliferation and suppress destructive overactivity byinflammatory and adipose cells, with production of many inflammogens.Whether amino phthalazinediones are actually a ligand for PPARγ, cansuppress tumor necrosis factor and resistin secretion in adipocytes andmacrophages, and increase secretion of adiponectin by adipocytes areunder investigation. Whether benzolated thiazolidinediones, like aminophthalazinediones, can bind to benzodiazepine receptors in mitochondriaand alter activity of ion channels and megapores in mitochondria arealso not presently known.

Since benzolated thiazolidinediones are very poor redox agents, it isnot likely that they directly modulate thiol redox status inmitochondrial voltage-dependent channels or in the permeability pores.In contrast, since amino phthalazinediones probably possess these dualdefensive functions, as a redox buffer in mitochondria and as a PPARactivator in the nucleus, amino phthalazinediones promise a better andmore complete therapy for all symptoms of metabolic syndrome.Combinational therapy with benzolated thiazolidinediones and aminophthalazinediones, plus thiols and other redox adjuvants, may be thetreatment of choice for prevention of downstream sequelae of metabolicsyndrome, such as hyperglycemia, hyper fatty acidemia, increased tumornecrosis factor and resistin levels, hypo-adiponectin-emia, hyper orhypo insulin-emia, impaired thiol redox status (hypo-glutathione andcysteine-emia), PPARγ inactivity, and mitochondrial energy uncouplingwith elevated H₂O₂, OHOO., and cytoplasmic cytochrome c.

Repeated monitoring of the above adipose hormones during treatments withbenzolated thiazolidinediones/amino phthalazinedione/thiol therapieswill be required to establish specific dosage and efficacy for eachindividual. Since with each individual, downstream sequelae of metabolicsyndrome, including insulin resistance with long-chain fatty acidpoisoning, vary greatly, dose adjustments according to individualresponses, as measured by the above adipokine markers, will be requiredfor optimum therapy.

EXAMPLE 16 Stress-Induced Phosphorylation Signaling andPhthalazinediones

The major survival and growth signaling pathways in some cells involvethe phosphorylation of epidermal growth factor receptor (EGFR),mitogen-activated protein kinases (MAPK), extracellular signal-regulatedkinases (ERK), phosphoinositol-3 kinase, protein kinase B, and inhibitor_(κ)B kinase (IKK), the kinase controlling NF_(κ)B activity, NF_(κ)Bbeing a major stress-induced transcription factor. The cell deathpathway is controlled by c-Jun N-terminal kinase (JNK), p38, and p53,another stress-induced transcription factor.

Oxidants such as H₂O₂ activate intracellular phosphorylation cascadesresponsible for cell survival and growth and for cell death viaapoptosis and necrosis (Wang et al., J. Biol. Chem. 275: 14624-14631,2000). Low doses of H₂O₂ directly and rapidly activate the survivalpathway, using primarily Akt, PI-3K, EGFR, and NF_(κ)B. The apoptoticfactors Bad and caspase 9 are also downregulated by low doses of H₂O₂.Higher doses of H₂O₂ or prolonged exposure to H₂O₂ activate the celldeath pathways involving JNK, p53, Bax, sphingomyelinase, caspases, andthe apoptosis signaling kinase AsK-1.

Thus, oxidants, much like the phthalazinediones of the invention,activate either cell survival or cell death pathways, depending ondosage. However, H₂O₂ is not a buffer and cannot maintain optimal redoxpotentials sufficient to maintain cell signaling and growth. H₂O₂ alsodoes not scavenge the excess reactive oxygen species produced byactivated cell growth pathways. The ability of phthalazinediones,especially amino phthalazinediones, to provide both oxidizing andreducing potential to mitochondria, peroxisomes, and cytoplasmicsignaling pathways makes these compounds an ideal in vivo redox buffercapable of dictating most cell fates.

In disease states where signal-induced cell death rates exceed cellgrowth rates—as in various neurodegenerative syndromes such asAlzheimer's disease, ataxia telangiectasia, Parkinson's disease,multiple system atrophy, or AIDS—or in disease states where autonomousgrowth signaling rates exceed cell death rates—as in cancers, ataxiatelangiectasia, trichothiodystrophy, or hyperinflammatorysyndromes—amino phthalazinediones dictate cell fates by buffering theaberrant cellular redox potentials up or down, both in the stressedpatient and in any invading or overactivated cell. The phthalazinedionesof the invention are likely to be therapeutically useful for modulatingaberrant phosphorylation signaling syndromes involved in cell growth anddeath.

EXAMPLE 17 Neuronal Overactivity and Amino Phthalazinediones

In Parkinson's disease, neurons of the subthalamic nucleus (STN) becomeimbalanced and discharge too much. This 4 Hz oscillatory overactivity inSTN neurons of patients with the classical symptoms ofparkinsonism—bradykinesia, rigidity, and tremor—is a major etiologicfactor in Parkinson's disease. Suppression of this oscillatory activityby intra-STN injection of various agents such as lidocaine and muscimol(a gamma aminobutyric acid-A receptor agonist) or chronic electrical(2V) stimulation promptly relieves these parkinsonian symptoms.

The cause of this 4 Hz overactivity in only a few STN neurons is notknown (Levy et al., Brain 124: 2105-2118, 2001; Luo et al., Science 298:425-429, 2002; Limousin et al., New England J. of Med. 339: 1105-1111,1998; Alvarez et al., Movement Disorders 16: 72-78, 2001). Thedownstream effects of STN overactivity in substantia nigra reticulata,globus pallidus, and motor thalamus are likely to be responsible formultiple movement disorders.

Since maintaining this excessive and imbalanced 4 Hz oscillationrequires increased energy expenditures, agents such as aminophthalazinediones, which can modulate thiol redox status, downregulatemitochondrial energy production, and gain access to the overactivatedSTN neurons, can potentially suppress the 4 Hz overactivity and therebysuppress and modulate the downstream network activities responsible forthe symptoms. Daily intraperitoneal injections of 200 μg of 4-sodiumamino phthalazinedione significantly delay the progress of the movementdisorder with paralysis induced by MOMU-LV-Ts1 virus in mice. Whetherthis is due to suppression of oscillatory activity in neurons,suppression of virus-induced astrocytic inflammatory responses, or bothis under investigation. Amino phthalazinediones should, however,suppress both the neuronal and astrocytic overactivity.

EXAMPLE 18 Multiple Chemical Sensitivity Syndrome

Multiple chemical sensitivity (MCS) is a decompensating syndrome inwhich individuals develop hypersensitivities to multiple environmentaltoxins, e.g., exhaust fumes from vehicles, factories, garbage dumps, orexplosives, perfumes, sulfur oxides, nitrous oxide, and cyclichydrocarbons produced by molds or plants. Inhalation of such toxinscauses symptoms like fatigue, weakness, loss of equilibrium, sensoryimpairments in smell, taste, hearing, vision, and sensation, cognitiveimpairments in memory, concentration, and motivation, and motor symptomsthat vary from muscle and bone wasting to athetoid, epileptiform, orfibrillary movements. External signs of premature aging, e.g., grayingor loss of hair, wrinkling of skin, bradykinesia, or signs of Syndrome X(type II diabetes, hypertension, hyperlipidemia, atrial fibrillation)are usually present at least to some extent.

The multiple incapacitating symptoms of MCS are rapidly induced byinhaling trace amounts of the noxious substance. Removal of the incitinginhalant prevents the acute symptomology but does not eliminate thehypersensitivity. The most characteristic symptom is an inability to“get up and go,” or lead a productive life. The symptoms may becomepermanent or persist for months unless the chronic exposure iseliminated. The inhaled toxins are thought to induce redox imbalances inthe naso-olfactory system and brain, and preliminary findings suggestthat the functional impairments in MCS are due to acute imbalances inNO/O₂ ⁻ and thiol redox potentials in the naso-olfactory system.

Studies also suggest that the neuromuscular and cognitive dysfunctionsin MCS may represent redox imbalances in pathways controllingneurotransmission. The constant neurotransmission in neurons, with itsrepetitive separation of charges, obligatorily produces large amounts ofreactive oxygen and nitrogen species. Under stress, these radicals mayaccumulate with pathologic results. The cell's major redox buffers forcontrolling these signaling radicals, e.g., reduced thiols such asglutathione, cysteine, and thioredoxins, are rapidly consumed underredox stresses such as anoxia, nutrient or ion deprivation, and aberrantpeptide accumulation.

The symptomatic redox imbalances may be corrected by appropriate redoxsupport to the impaired naso-olfactory system. Treatment with thiolredox support, i.e., inhaled or intramuscular glutathione, has beenpartially successful. Intranasal inhalation of a solution of 25 mg/ml ofa phthalazinedione of the invention and 12 mg/ml of sodium glutathionein isotonic NaCl four times per day for three days should remove theincapacitating weakness and the sensory and cognitive symptoms within 48hours. Inhalation of the reduced phthalazinedione, along with a reducedthiol, can quickly but temporarily rebalance the thiol redox imbalancein the patient's brain and quickly ameliorate some of the symptoms.Motor symptoms may be reversed by prolonged treatment with higher dosesof this combined redox therapy.

The foregoing material describes various aspects of the invention andhow it may be practiced. The description is not intended to beexhaustive of the many different embodiments of the invention. Althoughthe foregoing invention has been described in some detail by way ofillustration and example, to aid understanding, it will be readilyapparent to those of ordinary skill in the art, in light of theteachings of this invention, that certain changes and modifications maybe made to the invention without departing from the spirit or scope ofthe appended claims.

1-54. (canceled)
 55. A method for treating inflammatory conditionscomprising: administering to a subject in need thereof, atherapeutically effective amount of a phthalazinedione or apharmaceutically acceptable salt thereof, wherein the inflammatorycondition is selected from the group consisting of Parkinson's disease,multiple sclerosis, ulcer, arthritis, stroke, myocardial infarction,arteriosclerosis, atherosclerosis, inflammation of blood vessels thatcharacterize vascular disease in the heart and brain, thromboangiitisobliterans (Winiwarter-Buerger disease), dermatitis, eczema,postoperative complications, peritonitis, bronchitis, and pleurisy. 56.The method of claim 55 wherein the inflammatory condition is multiplesclerosis.
 57. The method of claim 55, wherein the inflammatorycondition is Parkinson's disease.
 58. The method of claim 55, whereinthe inflammatory condition is myocardial infarction.
 59. A method fortreating neurological disorders comprising: administering to a subjectin need thereof, a therapeutically effective amount of aphthalazinedione or a pharmaceutically acceptable salt thereof, whereinthe neurological disorder is selected from the group consisting ofmultiple sclerosis, dystonia, neurally mediated hypotension,fibromyalgia, reflex sympathetic dystrophy syndrome, myofascial painsyndrome and patellofemoral pain syndrome.
 60. The method of claim 59,wherein the neurological disorder is multiple sclerosis.
 61. The methodof claim 59, wherein the neurological disorder is fibromyalgia.
 62. Themethod of claim 59, wherein the neurological disorder is dystonia.
 63. Amethod for treating infectious conditions comprising: administering to asubject in need thereof, a therapeutically effective amount of aphthalazinedione or a pharmaceutically acceptable salt thereof, whereinthe infectious condition is selected from the group consisting of HIVinfection, acquired immunodeficiency disease, hepatitis, herpes, Lymedisease, dysentery, erysipelas, pneumonia, tuberculosis.
 64. The methodof claim 63, wherein the infectious condition is HIV infection.
 65. Themethod of claim 63, wherein the infectious condition is acquiredimmunodeficiency disease.
 66. The method of claim 63, wherein theinfectious condition is tuberculosis.
 67. The method of claim 63,wherein the infectious condition is pneumonia.
 68. A method for treatingimmune disorders comprising: administering to a subject in need thereof,a therapeutically effective amount of a phthalazinedione or apharmaceutically acceptable salt thereof, wherein the immune disorder isselected from the group consisting of leukemia, immune deficiencydiseases, transplant or graft rejection, allergic reactions, sinusconditions, eczema, asthma and diabetes mellitus.
 69. The method ofclaim 68, wherein the immune disorder is leukemia.
 70. The method ofclaim 68, wherein the immune disorder is an immune deficiency disease.71. A method for treating proliferative diseases comprising:administering to a subject in need thereof, a therapeutically effectiveamount of a phthalazinedione or a pharmaceutically acceptable saltthereof, wherein the proliferative disease is selected from the groupconsisting of leukemia, lymphoma, and myeloma, tumors, melanoma,carcinoma, sarcoma, prostatic hypertrophy or adenoma, atherosclerosis,angiogenesis, restenosis, proliferative diseases of the vascular systemor endometrium, and other syndromes of rapid cell proliferation orclonal expansion.
 72. The method of claim 71, wherein the proliferativedisease is melanoma.
 73. A method for treating senescence conditionscomprising: administering to a subject in need thereof, atherapeutically effective amount of a phthalazinedione or apharmaceutically acceptable salt thereof, wherein the senescencecondition is selected from the group consisting of Down syndrome,xeroderma pigmentosum, p53 overactivity, muscular dystrophy, metabolicsyndrome, and aging.
 74. The method of claim 73, wherein the senescencecondition is Down syndrome.